Structural Basis of the Proteolytic and Chaperone Activity of Chlamydia trachomatis CT441

Abstract

e Chlamydia trachomatis is the most prevalent cause of preventable blindness worldwide and a major reason for infectious infertility in females. Several bacterial factors have been implicated in the pathogenesis of C. trachomatis. Combining structural and mutational analysis, we have shown that the proteolytic function of CT441 depends on a conserved Ser/Lys/Gln catalytic triad and a functional substrate-binding site within a flexible PDZ (postsynaptic density of 95 kDa, discs large, and zonula occludens) domain. Previously, it has been suggested that CT441 is involved in modulating estrogen signaling responses of the host cell. Our results show that although in vitro CT441 exhibits proteolytic activity against SRAP1, a coactivator of estrogen receptor ␣, CT441-mediated SRAP1 degradation is not observed during the intracellular developmental cycle before host cells are lysed and infectious chlamydiae are released. Most compellingly, we have newly identified a chaperone activity of CT441, indicating a role of CT441 in prokaryotic protein quality control processes. Infections with the obligate intracellular pathogen Chlamydia trachomatis are among the most common sexually transmitted diseases worldwide, with approximately 1.5 million reported cases in the United States in 2012 (1). While most of the acute infections of the lower urogenital tract are asymptomatic and remain unrecognized by the affected people, ascending infections in females often result in severe chronic sequelae, such as pelvic inflammatory disease, ectopic pregnancy, and infertility (2). Despite its clinical relevance, many aspects of the underlying virulence mechanisms have not been elucidated so far.As for other pathogens, infectivity and the propensity to manipulate host immune responses largely depend on the repertoire of pathogenicity factors. The most extensively studied effector protein in Chlamydia research is CPAF (chlamydial protease-like activity factor), which has been reported to degrade a broad spectrum of host cell proteins (3). However, it has been shown that the observed degradation of many previously identified CPAF substrates is an artifact of the protein isolation process (4), and thus whether CPAF actively degrades host cell proteins during the intracellular developmental cycle is a controversial subject of discussion.A second chlamydial protease, designated CT441 for C. trachomatis, shares significant amino acid sequence similarity with tailspecific proteases (Tsps) from other species (e.g., 25% identity with Tsp from Escherichia coli) and was first proposed by Lad et al. to interfere with host antimicrobial and inflammatory responses (5, 6); however, in later reports, a role of CT441 and CPAF in the cleavage of NF-B during the chlamydial infection has been put into question (4, 7). Unique regions that show no similarities to any characterized domain are present at the N terminus of Tsp proteins. E. coli Tsp cleaves substrate proteins labeled with a Cterminal ssrA-encoded peptide tag (small stable RNA A) and is involved in ...

The Chlamydia trachomatis Protease CPAF Contains a Cryptic PDZ-Like Domain with Similarity to Human Cell Polarity and Tight Junction PDZ-Containing Proteins

“…This phenomenon is demonstrated by proteins in the tail-specific, DegP and HtrA protease families. In many of the S41 proteases, including DegP, HtrA2, HtrA3, and tail-specific proteases, secondary regulation of enzyme activity is achieved through communication between the catalytic serine protease domain and a nearby PDZ domain [ 35 , 54 , 55 ]. Because CPAF contains similarly arranged catalytic and PDZ domains, we hypothesize that the CPAF PDZ domain may be involved in enzyme activation or substrate recognition.…”

Production, crystallization and X-ray diffraction analysis of the protease CT441 fromChlamydia trachomatis

“…The asymmetric unit contains four CT441 molecules with a Matthews coefficient (Matthews, 1968) of 2.97 Å 3 Da À1 (estimated solvent content of 58.5%). Full structure determination, refinement and analysis of CT441 together with data on its biological function have been reported elsewhere (Kohlmann et al, 2015). …”

“…† R r.i.m. is the redundancy-independent merging R factor(Weiss & Hilgenfeld, 1997); part of the data-collection statistics in this table have been described in the Supporting Information ofKohlmann et al (2015).…”

“…The results presented by Borth et al (2010) indicate that CT441 is able to modulate the oestrogen- # 2015 International Union of Crystallography signalling pathway of the host cell by interacting with human SRAP1 (steroid receptor RNA activator protein 1), a coactivator of oestrogen receptor (ER ). We have previously reported the biochemical and structural characterization of CT441 (Kohlmann et al, 2015). Here, we report details of the production, purification, crystallization and X-ray diffraction analysis of an inactive variant of CT441.…”

The regulatory mechanism of fungal elicitor-induced secondary metabolite biosynthesis in medical plants

“…Chitin (β-1,4-linked polymer of N-acetylglucosamine) is an important structural component of fungal cell walls, and is also a well-known oligosaccharide elicitor of immune responses in plants (Kohlmann et al 2015). It also induces nodulation signaling in Lotus japonicas (Wang et al 2014).…”